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7LWOH Ca2+ coding and decoding strategies for the specification of neural and renal precursor cells during development

$XWKRU V Moreau, Marc; Neant, Isabelle; Webb, Sarah E.; Miller, Andrew L.; Riou, Jean-Francois; Leclerc, Catherine

6RXUFH Cell Calcium , v. 59, March 2016, p. 75-83

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'2, https://doi.org/10.1016/j.ceca.2015.12.003

3XEOLVKHU Elsevier

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This version is available at HKUST Institutional Repository via http://hdl.handle.net/1783.1/78363 If it is the author’s pre-published version, changes introduced as a result of publishing processes such as copy-editing and formatting may not be reflected in this document. For a definitive version of this work, please refer to the published version.

http://repository.ust.hk/ir/ This is the Pre-Published Version Moreau et al, review in Cell Calcium 2015

Titre : Ca2+ coding and decoding strategies for the specification of neural and renal progenitor cells during development. Authors : Marc Moreau, Isabelle Néant, Sarah E. Webb, Andrew L. Miller, Jean-François Riou, and Catherine Leclerc

Corresponding author: Catherine Leclerc

Introduction Calcium (Ca2+) signalling has long been reported to play a role in the early development of vertebrate embryos [1-3]. During embryogenesis, a rise in intracellular Ca2+ is known to be a widespread trigger for directing stem cells towards a specific tissue fate, but the precise Ca2+ signalling mechanisms involved in achieving these pleiotropic effects are still poorly understood. In this review, we compare the Ca2+ signalling pathways that are involved in regulating neural determination, which is the first step for both neural development, (neurogenesis) and kidney development (nephrogenesis). Figure 1 is a schematic representation of the Ca2+ signals that are known to be generated in the amphibian Xenopus laevis in the dorsal ectoderm during the process of neural induction, and in the dorso-lateral mesoderm during the specification of the embryonic kidney.

Early neurogenesis / neural induction It is important to bear in mind that although neural induction is considered to be similar (from the point of view of the morphological events and signalling pathways involved), for most if not all vertebrates, in reality only a few species have been studied in great detail. Indeed, much of the research on neural induction has been conducted with amphibian embryos. Traditionally, Xenopus sp. has been used as an animal model for investigating the inductive events and cellular mechanisms that occur during organogenesis because the embryos are easy to obtain in high numbers and are relatively large in size. Thus Xenopus embryos are an especially easy model system to use when performing microinjection and microsurgery. Neural induction occurs during gastrulation and the neural tissues, like the epidermal tissues, are derived from the ectoderm. During gastrulation in vertebrates, the ectodermal

1 This is the Pre-Published Version Moreau et al, review in Cell Calcium 2015 cells give rise to epidermal progenitors on the ventral side of the embryo and to neural progenitors in the dorsal side. This binary choice of cell fate during neural induction is controlled by complex mechanisms that involve both positive effectors (such as fibroblast growth factors, FGFs) and negative effectors (such as bone morphogenetic proteins, BMPs; Wingless/Int proteins, Wnts and Nodal) [4-6]. One of the key regulatory mechanisms involved in the conversion of ectoderm into neuroectoderm is the inhibition of the BMP pathway by noggin, chordin, and follistatin, which are factors secreted by the dorsal mesoderm. Following neural induction, the neuroectoderm develops into the neural plate, which consists of undifferentiated dividing neuroepithelial cells, and then later in development these cells exit the cell cycle and differentiate into neurons and glial cells. For the purposes of this article, however, we are limiting the topic of our review to that of neural induction, as this is the stage that specifically involves embryonic stem cells. With a combination of the bioluminescent Ca2+ reporter, aequorin, and a custom- designed luminescence imaging microscope, it has previously been shown that during

2+ 2+ gastrulation in amphibian embryos, a gradual elevation of intracellular Ca ([Ca ]i) and a series of superimposed rapid Ca2+ transients are generated exclusively in the dorsal ectoderm cells (the tissue where neural induction takes place). On the other hand, neither the Ca2+ elevation nor the distinct Ca2+ transients are generated in the ventral ectoderm cells, which are at the origin of the epidermis [7, 8]. The onset of this Ca2+ signalling activity occurs at the blastula stage, long before the start of gastrulation (i.e., before mesoderm invagination). These observations have been confirmed in Xenopus [9] and have also been demonstrated in the chick [10] suggesting that neural induction starts before gastrulation. In Xenopus embryos, the spontaneous Ca2+ transients are initially localized in the most anterior part of the dorsal ectoderm and the accumulation pattern of these Ca2+ transients over time correlates with the formation of the prospective neuroectoderm. In addition, as gastrulation proceeds, the Ca2+ transients increase both in number and intensity, so that they reach a peak activity by mid- gastrulation, a stage where neural determination is thought to have occurred [8]. Therefore, the Ca2+ transients observed in the dorsal ectoderm constitute the first directly visualized events linked to neural induction (Figure 1A, B).

The animal cap model

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In blastula stage Xenopus embryos, the animal cap constitutes the region around the animal pole, which is destined to form the ectoderm during normal development. This tissue retains it pluripotent nature and it can be easily dissected and maintained in simple culture medium. Upon exposure to specific inducers, however, the animal cap can differentiate into neural, mesodermal, or endodermal tissues. In this way, therefore, the cells of the animal cap have been described as being equivalent to mammalian embryonic stem cells [11]. Animal caps cultured in vitro have been shown to reproduce the in vivo induction of amphibian tissues and they are therefore considered to be a very useful tool for investigating the differentiation mechanisms that occur in normal embryonic development. Indeed, experiments with animal caps have demonstrated that in the amphibian embryo, Ca2+ is a necessary and sufficient to convert ectoderm into neuroectoderm [8]. In addition, animal caps have been induced to differentiate into neurons and glial cells following short term culture in the presence of caffeine, which triggers a rise of intracellular Ca2+ [12]. This ex vivo assay has also been used to further characterize the Ca2+ signalling pathways that lead to the expression of neural specific genes.

Implication of calcium channels. The ability of ectoderm cells to be induced to differentiate into neural tissue (called neural competence) [13, 14], is acquired shortly before gastrulation and is then lost during the late gastrula stages. In amphibians, neural competence is associated with the expression of

2+ functional CaV1.2 channels (i.e., L-type voltage-dependent Ca channels) in the plasma membrane [15, 16]. The highest density of CaV1.2 channels is reached at mid-gastrula, when neural competence of the ectoderm is optimal. There is subsequently a decrease in the density of CaV1.2 channels, which occurs simultaneously with the normal loss of competence, at the end of gastrulation. This temporal pattern of CaV1.2 channel expression correlates with the dynamic pattern of Ca2+ transients.

When the function of CaV1.2 channels was inhibited by treatment with specific antagonists during gastrulation, there was a concomitant inhibition in the generation of Ca2+ transients and a decrease in the resting level of intracellular Ca2+, which suggests that the Ca2+ transients might be generated via the activation of CaV1.2 channels [7, 8]. In addition, the inhibition of these Ca2+ transients induced a downregulation of at least two of the early neural genes (i.e., zic3 and geminin) as well as severe abnormalities in the anterior nervous system.

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The most apparent defects were observed in the head including a reduction in the size or total absence of eyes, as well as a lack of melanophores [8, 17]. Thus, in the naïve ectoderm cells

2+ of Xenopus, an influx of Ca through CaV1.2 channels is likely to be the main component of

2+ the signalling pathway that induces the changes in [Ca ]i observed both in vivo and ex vivo during neural induction [18]. However, It should be noted that members of the TRP (transient receptor potential) channel family, particularly TRPC1, are also likely to be involved [19]. Acquisition of neural fate in amphibians therefore appears to require the expression of functional CaV1.2 channels in the ectoderm. This finding, however, prompted several new

2+ questions: (1) Given that CaV1.2 channels are voltage-operated Ca channels, what is the mechanism by which these channels are specifically activated in the dorsal ectoderm during the process of neural induction? (2) What are the genes activated downstream of the Ca2+ signals? (3) Are the Ca2+ signals that are involved in neural induction, conserved among vertebrates?

Genes controlled by Ca2+ Amphibian embryos and animal caps are both useful for screening the downstream target genes, which are activated by Ca2+ signalling during neural induction [20]. In particular, it has been shown that Ca2+ controls the expression of the immediate early gene c-fos [21] as well as that of two other transcription factors: XlPou2 and zic3 [8]. Whereas Fos is a ubiquitous transcription factor, XlPou2 and zic3 are primary neural regulators, which are specific to neural determination [22, 23]. Furthermore, it has been shown that the upregulation of XlPou2 in response to noggin in animal caps, and the expression of zic3 in intact embryos, both require the presence of functional CaV1.2 channels [8]. Keller explants (i.e., a two dimensional explant system comprised of prospective neuroectoderm and dorsal mesoderm [24]), have also been used to demonstrate that the accumulated pattern of Ca2+ transients correlates with the expression of zic3. In addition,

2+ nifedipine, a CaV1.2 channel antagonist, was shown to block the Ca transients and reduce the level of zic3 expression [25]. These results suggest that one function of the localized

2+ increase in [Ca ]i, which occurs in the dorsal ectoderm during neural induction, might be to activate genes with proneural activity in distinct regions of the embryo. To identify new Ca2+ target genes involved in neural induction, a subtractive cDNA library was constructed between untreated (i.e., ectodermal cells fated to become epidermis)

This is the Pre-Published Version Moreau et al, review in Cell Calcium 2015 and caffeine-treated animal caps (i.e., ectodermal cells fated to become neural tissue) [26].

2+ Caffeine triggers neural induction via an increase in [Ca ]i [27], and thus allows the differential isolation of the earliest Ca2+-dependent genes involved in neural determination [26]. Approximately 30 clones were found to selectively hybridize to the neural-subtracted probe and not to the epidermis-subtracted probe. Among these clones, we identified xmlp, prmt1b, xId3 and p54nrb [29]. Xmlp encodes a MARCKS-like protein, which is a substrate for protein kinase C (PKC) [26, 28]; prmt1b is the Xenopus homologue of the mammalian arginine methyltransferase PRMT1 gene [29]; xId3 is an HLH factor that acts as dominant negative inhibitor of bHLH transcription factors [30]; and p54nrb encodes an RRM-domain protein characteristic of RNA-binding proteins and it has been implicated in pre-mRNA splicing steps [31]. The spatio-temporal expression pattern of these genes was shown to be restricted to the neural territories and their expression was triggered following an inhibition of BMP signalling by noggin. Interestingly, expression of xmlp and prmt1b occurs as an early response to the increase in Ca2+ and does not require de novo protein synthesis. In addition, the early expression of prmt1b during the gastrula stage in Xenopus embryos was shown to occur via a Ca2+-dependent mechanism mediated by the activation of CaV1.2 channels, and a functional analysis of this gene demonstrated that it is required for neural induction. When prmt1b was over-expressed in the neural territories, the expression of the neural precursor gene zic3 was induced. Conversely, the loss of function of prmt1b inhibited the expression of zic3 and impaired anterior neural development in the whole embryo [29]. An identical phenotype was also obtained when embryos were treated with CaV1.2 channel antagonists [8]. Therefore, during neural induction, prmt1b provides a

2+ direct link between the increase in [Ca ]i, and downstream events such as the regulation of gene expression. A likely mechanism might involve the methylation of early neural specific genes. Accumulating evidence suggests that Ca2+ is also an important positive regulator of neural induction in other vertebrates as well as in some invertebrate species. In intact zebrafish (Danio rerio) embryos, for example, localized and transient elevations of intracellular Ca2+ are generated in the ventral side of the blastoderm margin during early gastrulation. As gastrulation proceeds, additional localized Ca2+ transients are generated around the blastoderm margin, and these signals are the sources for long-range propagating Ca2+ waves that either circumnavigate the blastoderm margin or propagate along the forming embryonic

5 This is the Pre-Published Version Moreau et al, review in Cell Calcium 2015 axis [32]. By the end of gastrulation, the periodic Ca2+ waves originate exclusively from the embryonic shield (or organizer) in the dorsal region of the embryo [32]. These distinct Ca2+ signalling patterns correlate both temporally and spatially with neural induction. Furthermore, when the intracellular Ca2+ signals generated during early stages of zebrafish development were buffered with BAPTA (a Ca2+ chelator), this lead to embryos with small eyes [33] and with disrupted motoneuron development [34]. In chick embryos, a transmembrane Ca2+ channel facilitator called calfacilitin, was identified that increases the Ca2+ flux by generating a larger window current and slowing the inactivation of the CaV1.2 channel [35]. Calfacilitin was shown to be induced by FGF, and it binds to, and is co-expressed with, the CaV1.2 channel in the embryo. It was also shown to be required for neural induction and for the expression of the neural plate specifiers, geminin and sox2, as well as for neural plate formation. Finally, using a genetically encoded Ca2+ indicator in Ciona intestinalis embryos, multiple Ca2+ transients were observed throughout the developing neural plate [36]. In addition (and similar to in amphibians), treatment of Ciona embryos with Ca2+ blockers during gastrula and neurula stages resulted in a disruption in the development of the anterior neural plate [36].

Ca2+ early target genes is not restricted to neural tissue Among the clones isolated from the original subtractive cDNA library [27], the expression patterns of three additional early Ca2+-target genes were identified; fus (fused in sarcoma), brd3 (bromodomain containing 3) and wdr5 (WD repeat domain 5). All three genes represent potential regulators of the transcriptional machinery in early Xenopus development. Furthermore, in contrast to prmt1b and p54nrb, the expression domains of fus, brd3 and wdr5 are not just restricted to the neural territories but are also present in other tissues, principally in the pronephros. Similarly, xmlp which was also previously isolated in the subtractive screen, is also expressed in pronephric territories [28]. We therefore propose that these Ca2+ target genes might control different aspects of the transcriptional machinery during organogenesis and in this way regulate the specification of neural and/or renal progenitors. In the next section, the role of Ca2+ in the specification of the kidney field is discussed.

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Nephrogenesis in Amphibians :Specification of the renal field in embryos. The first kidney to form in embryos of all vertebrate species is the pronephros, and this arises de novo from cells in the intermediate mesoderm. Specification of the pronephric tissue field can first be identified by the end of gastrulation, at the early neurula stage. This embryonic kidney is subsequently replaced by a mesonephros and then (in birds and mammals) by a metanephros [37]. In Xenopus laevis larvae there are two pronephroi, each of which consists of just one nephron (the functional unit of the kidney), which forms between the paraxial mesoderm and the lateral plate, just behind the head. The pronephros is comprised of a large glomus, which filters waste into the coelom; a coiled tubule, which collects the filtrate via three ciliated nephrostomes, and a pronephric duct, which transports the waste to the cloaca [38]. The structure of the nephron and the molecular mechanisms involved in its formation are evolutionary conserved [39-41]. Because of its simple organization and rapid development (i.e., the pronephros is functional within just 2 days), the amphibian pronephros provides an easy model to study how pluripotent mesodermal cells are committed to a renal fate [42]. Anotheradvantage of the amphibian model is that (as described previously) the use of the animal cap assay provides a simple and effective 2D model for investigating complex differentiation events and mechanisms [11]. Indeed, co-treatment of animal cap ectodermal cells with activin A (a mesodermal inducer) and retinoic acid leads to the formation of a functional pronephros, composed of the three lineages: the glomerulus, pronephric tubule and duct [43]. The ability to obtain differentiated pronephros from animal caps incubated with retinoic acid and activin A has resulted in the identification of a link between Ca2+ signalling and gene expression in pronephric development.

Early genes involved in the specification of the embryonic kidney Several genes, which encode transcription factors have been identified in the mouse as playing an important role during renal development [39]. Orthologues of these same have also been shown to be expressed in the lateral mesoderm at the onset of neurulation in amphibians [39]. They include pax8, lhx1, osr1 and osr2 [44, 45]. Loss of function of osr1 and osr2 [45] and lhx1 [46] impair the development of the pronephros, while overexpression of pax8 and/or lhx1 results in the development of ectopic and enlarged pronephroi [44]. The mesodermal territory where the expression of pax8 and lhx1 overlap has been defined as the pronephric field or kidney field (KF).

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The mechanisms controlling the emergence of the KF from the mesodermal layer in gastrula stage embryos are still only partially understood. However, it is known that FGF signalling needs to be down-regulated to allow mesodermal cells to adopt a pronephric fate [47, 48], and that the Wnt pathway is a positive regulator of KF specification. Wnt-11b induces pronephric structures in unspecified lateral mesoderm explants, and also acts as a potential inducer [49]. In addition, retinoic acid (RA) signalling is required during gastrulation for specification of the KF; when this signalling pathway is disrupted then there is a loss of pax8 and lhx1 expression in the KF, and a loss of glomus and tubule development at later stages [50].

Ca2+ signalling and the specification of the renal progenitors Evidence indicates that one or more Ca2+ signalling pathway might also be a major regulator of KF specification. For example, in Xenopus embryos at the early tailbud stage, ndrg1 (a member of the N-myc downstream-regulated gene family), is expressed in the presumptive pronephric territory and depletion of ndrg1 results in a failure of pronephros development [51]. Its expression is ubiquitous in tissues and it can be modulated by numerous agents including hypoxia and RA [52], as well as via an increase in intracellular Ca2+ through a mechanism independent of the cellular stress pathway [53]. The Xenopus homologue of the senescence marker protein-30 (SMP-30), is selectively expressed in the pronephric tubules from the late tadpole stage and in animal caps treated with activin A and retinoic acid [54]. SMP-30 (or regucalcin [55]), is a Ca2+-binding protein that is highly conserved in vertebrates. It has been shown to participate in the regulation of cytosolic Ca2+ homeostasis by increasing the activity of a Ca2+-pumping ATPase in the basolateral membranes isolated from rat kidney cortex [56]. Heat shock 70-kDa protein 5 (Hspa5), belongs to the heat shock protein 70 kDa family; it plays a role in protein folding and Ca2+ homeostasis as well as being a regulator of the endoplasmic reticulum (ER) stress response. Hspa5 plays an essential role in pronephros formation, mediated in part through RA signalling [57]. Similarly, Hspa9 (Heat shock 70kDa protein 9) is highly expressed in the developing pronephros territory of Xenopus embryos and it is known to control tubule formation. In addition, the protein it encodes, Hspa9, is known to be a Ca2+-binding protein and mitochondrial chaperone, [58].

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The presence of these various genes and the proteins they encode and their requirement in the specification of the KF suggests that changes in intracellular Ca2+ might also be important for this process. Indeed, aequorin-imaging revealed that in intact embryos, Ca2+ transients occur in the lateral mesoderm during kidney tubule formation (Figure 1C, D). These Ca2+ transients were distributed spatially within the area where the pronephric tubule subsequently differentiated, and they correlated with the expression pattern of the pronephric tubule marker, smp-30. In addition, inhibition of these transients with a photo- activatable Ca2+ chelator during the late gastrula or mid-neurula stages resulted in the defective development of the tubule [59]. In animal cap cells treated with RA and activin A to induce pronephric tubule formation, it was the former alone that stimulated the generation of Ca2+ transients. Furthermore, incubation of activin A-treated ectoderm with caffeine or ionomycin, which increase the concentration of intracellular Ca2+, can trigger tubule differentiation, indicating that Ca2+ signalling might be sufficient to regulate this process. Conversely, tubule formation can be inhibited by incubating ectodermal tissue with the Ca2+ chelator, BAPTA-AM or with lanthanum chloride (La3+) prior to treatment with activin A and RA. These data indicate that Ca2+ might be a necessary signal in the process of tubulogenesis both ex vivo and in intact embryos [59].

TRP Channels are involved in the generation of the Ca2+ signals observed in the renal progenitors The inhibitory effect of La3+ (a potent antagonist of many members of the transient receptor potential (TRP) channel family [60]), on tubulogenesis strongly suggests that TRP channels are involved in the process [59]. A likely candidate is the TRPP1/TRPP2 (or polycystin- 1/polycystin-2) complex [61]. The precise functions of TRPP1 and TRPP2 (transient receptor potential polycystin 1 and 2) are not completely understood, but there is growing evidence that TRPP2 might function as a Ca2+-regulated/Ca2+ permeable cationic channel and that TRPP1 is a regulator or sensor of this channel [62]. TRPP1 has previously been shown to be expressed in the presumptive pronephric territory in intact Xenopus embryos and in animal caps treated with activin A and RA [63]. TRPP2 is known to be localised in various different subcellular compartments. These include the basolateral plasma membrane of epithelial cells [64]; the endoplasmic reticulum

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(ER) where it may operate as a Ca2+-release channel [65], and the primary cilia where it is thought to participate in mechanosensation [66]. The function of TRPP2 in primary cilia has been linked to the formation of kidney cysts in vertebrates. TRPP2 is encoded by the PKD2 gene and in humans, mutations in PKD2 are responsible for autosomal dominant polycystic kidney disease (ADPKD) [67]. In addition, morpholino (MO)-based knock-down of pkd2 has been shown to result in the formation of pronephric cysts in zebrafish [68, 69], and in the formation of severe oedema and dilated pronephric tubules in Xenopus [70]. In Xenopus, the role of TRPP2-dependent Ca2+ signalling in the specification of lateral mesoderm into the KF has recently been addressed [71]. Whereas MO-based knock down of pkd2 resulted in a significant inhibition in pax8 expression in the KF, the expression of other KF genes such as lhx1 and osr1 and 2 was unaffected. Furthermore, MO-mediated knock down of pkd2 resulted in a dramatic decrease in the number of Ca2+ transients in KF explants. In addition, the use of a photo-inducible Ca2+ chelator to inhibit Ca2+ signalling from the midgastrula stage, also resulted in the reduced expression of pax8 in the KF. This particular function of TRPP2 was shown to be associated with the localization of the channel to the plasma membrane and not to the primary cilium. Together, these results indicate that TRPP2 might be directly involved in the generation of Ca2+ signalling and that it acts very early during development to specifically control the expression of pax8, a master gene regulator of pronephric kidney development [71]. Since pkd2 knockdown specifically affected the expression of pax8 and not the other genes involved in pronephros development, it is possible that Ca2+ signalling might be an intermediate step in the general mechanisms (i.e., RA and Wnt signalling pathways) that control the emergence of the KF within the lateral mesoderm. Furthermore, this Ca2+ signalling pathway is likely regulated by TRPP2, and it acts upstream of the activation and/or maintenance of pax8 expression in the KF. Most recently, we have further investigated the mechanism by which RA might affect the expression of pax8. We showed that RA does not affect the expression of pkd2 mRNA in the lateral mesoderm cells. In addition, using total internal reflection fluorescence (TIRF) microscopy we demonstrated that disruption of RA signalling by Cyp26, (a critical enzyme in the degradation of RA), inhibited the incorporation of TRPP2 channels into the plasma membrane. A hypothetical model to illustrate how TRPP2-dependent Ca2+ signalling might control pax8 expression in the kidney field during Xenopus gastrulation is shown in Figure 2.

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How might Ca2+ activate genes that are involved in development? Ca2+ might control gene expression either indirectly, via changes in the transactivating properties of transcription factors following the activation of Ca2+-dependent kinases and/or phosphatases [72-75], or directly via nuclear Ca2+ sensors [76]. During the specification of neural progenitors in amphibians, prmt1b has been shown to bind directly to Ca2+ target without requiring de novo protein synthesis [29]. To date, DREAM (i.e., Downstream Regulatory Element (DRE) Antagonist Modulator), is the only Ca2+ sensor known to bind specifically to DNA and to directly regulate transcription in a Ca2+- dependent manner [77]. DREAM (also called calsenilin, KChIP-3 and Kcnip3), is an EF-hand Ca2+ binding protein, and it is one of four Kcnip proteins that are known to regulate the membrane expression and gating of Kv4 potassium channels [78]. The affinity of Kcnip3 to the DRE binding site is modulated by the Ca2+ occupancy of its EF-hand sites. In addition, according to various cellular models, Kcnip proteins have been shown to positively or negatively affect cell proliferation by controlling cell-cycle regulators [76, 79-81].

Kcnips in neurogenesis Since little information was available about the role of the Kcnip proteins during neural induction in vertebrates, we investigated the temporal expression pattern of the various kcnip mRNAs in Xenopus laevis [82]. Among the four kcnips only Kcnip1 was expressed at all developmental stages, from fertilized egg (stage 1) to the tadpole (stage 46) stages. In contrast, the transcripts for, kcnip2, kcnip3 and kcnip4 were expressed at stages much later than neural induction. Thus, from a temporal expression pattern point of view, only kcnip1 was compatible with the neural induction stage in Xenopus laevis. Kcnip1 protein is known to possess Ca2+-dependent DRE-binding activity. Furthermore, loss-of-function of kcnip1 has been shown to increase the number of proliferating neural precursors; expand the neural plate (as seen by the enlargement of the anterior expression domain of early neural genes prmt1b, zic3 and sox2); inhibit epidermis and neural crest formation as shown by the reduction in expression of k81 and sox8, respectively; and block expression of n-tub, a marker of neuronal differentiation. These data therefore suggest that extension of the neural plate might be controlled by kcnip1 via transcriptional repression of early neural genes such as prmt1b involved in neural fate specification [29], and/or of sox2 required to maintain neural progenitor identity.

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Before gastrulation, the ectodermal cells are multipotent and can give rise either to epidermis or neural tissue. The mechanisms that govern the choice of neural fate require the inhibition of BMP pathway, as well as the activation of the FGF and Ca2+ signalling pathways [83]. In situ hybridization analysis showed that kcnip1 is expressed in both the dorsal and ventral ectoderm at the early gastrula stage. However, the accumulative pattern of Ca2+ transients observed exclusively in the dorsal ectoderm during gastrulation, strictly marks the neural plate [8, 84, 85] and correlates, at the neurula stage, with the expression of kcnip1. This suggests that the Ca2+ activities observed during neural induction in the dorsal ectoderm might activate the Kcnip1 Ca2+-dependent repression of the early neural genes specifically in this territory.

Kcnips in nephrogenesis There is evidence to suggest that Ca2+ might also directly regulate the expression of genes involved in nephrogenesis. Several of the genes that were isolated during the Ca2+ differential screen, such as xmlp, brd3, wdr5 and fus [26], are also expressed in the pronephric territory [86] and have putative DRE sites. In amphibians, the expression of pax8, a master gene in the establishment of the kidney field, depends on intracellular Ca2+ signalling [71]. However, the mechanism by which Ca2+ regulates pax8 transcription is still unknown. It has been suggested that in mouse thyroid cells, Ca2+ might exert direct control on pax8 transcription by releasing DREAM-dependent pax8 repression. Indeed, functional DRE sites have been located in the mouse pax8 promoter at positions 162 and 383 upstream of the coding sequence [87]. One potential DRE site (i.e., ctgctGTCAagatca) is present 287 nucleotides upstream of the pax8 coding sequence in Xenopus, and kcnip1 expression is prominent in the pronephric tubule [82]. Of note, Kcnip3 is expressed in, and pax8 has been shown to control nephron differentiation and branching morphogenesis of, the metanephros in mammals [76, 88, 89]. Altogether, these results suggest that kcnip family members may play essential roles in the control of pax8 expression during the specification of the pronephric progenitors as well as during the development of metanephric kidney.

Conclusions

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In this review, we have summarized more than 10 years of research on the role played by Ca2+ in the specification of neural and renal progenitors in amphibians. The different functions of Ca2+ signalling during these two events illustrate the versatility of Ca2+ as a second messenger. In addition, the duality of signalling pathways is not uncommon during development; indeed, identical signalling pathways, including those involving BMP, Wnt, FGF and GDNF are implicated during both neural and kidney development in Xenopus embryos [6, 90]. The requirement for Ca2+ signalling has also been demonstrated during the early phases of both neural and pronephros development in amphibians [59, 71, 84]. We have highlighted the role played by Ca2+ influx and by Ca2+ transients in the determination and differentiation of pools of neural or renal progenitors. Several regulatory events have shown to be modulated by Ca2+, including both short term effects and longer- lasting modifications, which ultimately lead to altered gene expression. Specific Ca2+ signals are decoded by different Ca2+ sensors, each having its distinct binding properties (e.g., affinity, conformational changes), which allow for fine tuning of particular Ca2+ signalling pathways. Among the Ca2+ sensors, those that contain EF-hand Ca2+-binding sites are known to play essential roles. For example, EF-hand proteins such as calmodulin (CaM) and CaM kinase (CaMK) are known to play a role in early neurogenesis (review in [91]), whereas calbindin- D28K and S100E are involved during neuronal differentiation and neurite outgrowth later on in development [92, 93]. With regards to kidney development, Ca2+ signalling has largely been studied through the characterization of Ca2+-binding proteins such as the senescence marker protein-30 (SMP30), calretinin, calbindin 1 and 2, parvalbumin, S100E and annexin IV [94, 95]. In this review, we also described a mechanism whereby the transcriptional control of gene expression during neurogenesis and nephrogenesis might be directly controlled by Ca2+ signalling. This mechanism involves members of the Kcnip family such that a change in their binding properties to specific DNA sites is a result of Ca2+ binding to EF-hand motifs. This role for Ca2+ signalling in transcription isn’t simply limited to genes involved in neurogenesis and nephrogenesis, however, as Kcnip has also been shown to participate in the regulation of Ca2+ homeostasis. In cardiac myocytes, for example, the Ca2+-calmodulin-dependent protein kinase II (CaMKII) has been demonstrated to down-regulate the expression of CACNA1C by controlling the nuclear translocation of the transcription repressor Kcnip3/DREAM [96]. In the nucleus, Kcnip3 binds ƚŽĂZƐŝƚĞĂƚƉŽƐŝƚŝŽŶоϱϭϭŝŶƚŚĞCACNA1C promoter, repressing the transcription of the Cav1.2 channels. Under these conditions, the down-regulation of the

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2+ CaV1.2 channel through the Ca /CaMKII/Kcnip3 cascade is a physiological feedback

2+ mechanism enabling the cells to adjust the Ca influx via CaV1.2 channels. It has been shown that in transgenic mice that overexpress a Ca2+-insensitive dominant active Kcnip3/DREAM mutant, there is a significant reduction in CACNA1C mRNA levels in both the heart and cerebral cortex [97]. These data therefore further support the idea of a self-regulatory loop by which Ca2+ might regulate Ca2+ homeostasis. Even though information is accumulating all the time with regards to the mechanisms involved in neurogenesis and nephrogenesis, there is still a lot to learn about both developmental processes. One of the main goals will be to determine how, and via which combination of specific Ca2+ toolkit elements [98], Ca2+ might regulate specific events in neural and renal progenitor cells in order to drive the overall development of these two quite distinct organ systems.

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Acknowledgements In France, this work was supported by the Centre National de la Recherche Scientifique (CNRS), and in Hong Kong, our research is supported by RGC awards: HKUST662113, 16101714 and 16100115; an ANR/RGC award: A-HKUST601/13, and TRS award: T13-706/11- 1.

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Figure 1 Schematic representation of the various Ca2+ events that occur during induction of the neural and renal progenitors in Xenopus laevis embryos. (A) Developmental time scale showing both the time post-fertilization (in hours post fertilization; hpf) and stage number during gastrulation and neurulation. (Ba) Dorsal ectodermal cells (in green) are induced to follow a neural fate during the process of neural induction and they give rise to neural progenitors (in yellow). (Bb) The graph is a schematic representation of the Ca2+ signals generated in the dorsal ectoderm from stage 8 to the end of gastrulation (stage 12). Two types of Ca2+ signals have been described during this process such that a series of Ca2+ transients (represented by the vertical lines), is superimposed on a slow rise in Ca2+. These Ca2+ signals have been shown to be due to influx through the Cav1.2 channel and (perhaps also) through TRPC1 channels, and they are required to control the expression of early neural genes such as prmt1b and zic3, which are necessary for neural induction. Inset: Schematic representation of early gastrula (stage 10) and late gastrula (stage 12) embryos from a dorsal view. The dashed line and circle on the stage 10 and stage 12 embryos, respectively, shows the position of the blastopore lip and yolk plug, respectively; dotted area represents the dorsal ectoderm and red dots the localisation of the Ca2+ transients. The onset of Ca2+ transients starts in the most anterior part of the dorsal ectoderm. Additional details regarding this mechanism are described in the text and [84]. (C) Developmental time scale showing the time post- fertilization (in hpf) and stage number from the end of gastrulation to a 2-day old larva. (Da) Lateral mesodermal cells (in light yellow) are induced to develop into renal progenitors via the process of pronephric induction (in brown). (Db) The graph is a schematic representation of the Ca2+ signals recorded in the lateral mesodermal cells from stage 12 to stage 35/36, when pronephros formation is complete. Similar to the Ca2+ signals generated during neural induction (see panel Bb), during pronephric induction a series of Ca2+ transients (represented by the vertical lines), is superimposed on a slow rise in Ca2+. These Ca2+ signals are due to influx through TRPP2 channels and are required to control the expression of pax8, which is known to be required for pronephric induction. Inset: Schematic representation of a late gastrula (stage 12) embryo from a dorsal view. The dashed circle shows the position of the yolk plug; the two dotted areas represent the presumptive kidney field and the red dots indicate the localisation of the Ca2+ transients. Additional details regarding this mechanism are described in the text and [59, 71].

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Figure 2 Schematic illustration to show the mechanism by retinoic acid might act via TRPP2- dependent Ca2+ signalling to control pax8 expression in the kidney field. This model is based on previously published results [50, 59, 71]. We propose that retinoic acid (RA) can control the incorporation of TRPP2 channel into plasma membrane (1), which leads to an influx of Ca2+

2+ 2+ through these channels (2). The resulting increase in intracellular Ca ([Ca ]i) in the kidney field controls pax8 expression (3). The mechanism by which Ca2+ regulates pax8 transcription is still unknown.

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